Members of the genus Salmonella are ubiquitous pathogens found in humans and livestock, as well as in wild animals, reptiles, birds, insects and in the environment. Salmonella causes diseases such as gastroenteritis and enteric fever in both humans and animals. The World Health Organization (WHO) estimated that in the year 1980 Salmonella caused more than one billion cases of acute diarrhea in children under five years of age in developing countries, and that five million of these children died. (Garthright W. E., Archer D. L., Kvenberg J. E. (1988) Estimates of Incidence and Costs of Intestinal Infectious Diseases in United States. Public-Health Rep 103:107-115.) The world-wide incidents of Salmonellosis (food poisoning of humans infected with Salmonella) has been increasing during the 1980's and 1990's (Todd E. (1990) Epidemiology of Food Borne Illness: North America. Lancet 336:788-790; and Cooke E. M. (1990) Epidemiology of Food Borne Illness: UK. Lancet 336:790-793.) The costs of food borne gastroenteritis in the U.S. are astonishing. There are five different estimates summarized by Todd (1990) which range from 4.8 to 23 billion dollars per year; Salmonella infections are a major component of these costs.
The incidence of Salmonellosis has changed dramatically in the last few years. The incidence of typhoid fever caused by S. typhi has been greatly reduced in the developed world in the last 50 years. However, it is still a major disease in developing countries. At the same time, there has been a marked increase in the incidence of non-typhoid Salmonellosis in the United States and worldwide. Although non-typhoid Salmonellosis is often a self-limiting event, including symptoms like non-bloody diarrheal stools, nausea, abdominal pain, and vomiting, it can proceed to more serious complications in patients with underlying diseases such as HIV-AIDS, sickle cell anemia, liver and gall bladder diseases. S. typhimurium most commonly causes infections and disease in both humans and animals. S. typhi only infects humans, and causes the dreadful illness typhoid fever. Typhoid fever kills about 10% of all people infected.
Infection by non-typhoid Salmonella is usually caused by contaminated food or via animals or pets. Infection by S. typhi is frequently caused by food mishandling or by carriers, who may themselves appear healthy. Such apparently healthy carriers are referred to as "asymptomatic carriers" of S. typhi. Development into an asymptomatic carrier state is well known in the Salmonella infection progression. When employed as food handlers, chronic asymptomatic carriers can pose a serious threat to the public health. A classic example is Typhoid Mary, a New York City cook who spread typhoid fever to many people before she was apprehended and imprisoned for life. (Salyers A. A., and Whitt D. D. (1994) In Bacterial Pathogenesis, a Molecular Approach A.S.M. Press, Washington, D.C.)
It would be desirable to create a monitoring system for virulent strains of Salmonella utilizing modern comparative molecular genomic approaches.
Various current typing techniques that can distinguish between strains of microorganisms can be divided into two major categories: those based on phenotypic characteristics and those based on genotypic characteristics. The former techniques include isozyme electrophoresis, whole-cell protein profiling (Senior B. W., and Voros S. (1990) Protein Profile Typing--a New Method of Typing Morganella morganii Strains J. Medical Microbiol. 33:259-64), sugar metabolism profiling, total fatty acids profiling (Guerrant G., Lambert M. A., Moss C. W. (1982) Analysis of Short-Chain Acids From Anaerobic Bacteria by High-Performance Liquid Chromatography J. Clin. Microbiol. 16:355-360), and various immunoblotting techniques (Persing D., Smith T. F., Tenover F. C., White J. (eds.) (1993) Diagnostic Molecular Microbiology American Society For Microbiology, Washington, D.C.).
Typing techniques based on genotype characteristics include DNA-DNA hybridization, restriction enzyme analysis (RFLP), ribotyping (Bingen E. H., Denamur E., Elion J. (1994) Use of Ribotyping in Epidemiological Surveillance of Nosocomial Outbreaks Clin. Microbiol. Rev. 7:311-327), plasma profiling (Grattard F., Pozzetto B., Berthelot P., Rayet I., Ros A., Lauras B., Gaudian O. G. (1994) Arbitrarily Primed PCR, Ribotyping, and Plasmid Pattern Analysis Applied to Investigation of a Nosocomial Outbreak Due to Enterobacter cloacae in a Neonatal Intensive Care Unit J. Clin. Microbiol. 32:596-602), DNA fingerprinting by Arbitrarily Primed PCR (APPCR), (Welsh J., McClelland M. (1990) Fingerprinting Genomes Usiniz PCR with Arbitrary Primers Nucleic Acids, Res. 18:7213-7218), random amplified polymorphic DNA (RAPDs) (Williams J. G. K., Kubelik A. R., Livak K. J., Rafiliski J. A., Tingey S. V. (1990) DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers Nucleic Acids, Res. 18:6531-6535), and rep-PCR (Versalovic J., Koeuth T., Lupski J. R. (1991) Distribution of Repetitive DNA Sequences in Eubacteria and Application to Fingerprinting of Bacterial Genomes Nucleic Acids, Res. 19:6823-6831).
Among the currently available commercial diagnostic assays for Salmonella are miniaturized biochemical tests utilizing nucleic acid-based assays (Aabo S., Andersen J. K., Olsen J. E. (1995) Research Note: Detection of Salmonella in Minced Meat by the Polymerase Chain Reaction Method Lett. Appl. Microbiol. 21:180-2); Lin C. K., Tsen H. Y. (1995) Development and Evaluation of Two Novel Oligonucleotide Probes Based on 16S rRNA Sequence in the Identification of Salmonella in Foods J. Applied Bacteriol. 78:507-520); and Olsen J. E., Aabo S., Rasmussen O. F., Rossen L., (1995) Oligonucleotide Probes Specific for the Genus Salmonella and for Salmonella typhimurium Lett. Appl. Microbiol. 20:160-163), and antibody-based assays (Feng P. (1992) Commercial Assay Systems for Detecting Food Borne Salmonella J. Food Prot. 56:927).
Regardless of whether a phenotypically-based typing method is used or a genotypic-based typing method is used, a relatively pure culture of microorganisms is required. Currently available Salmonella typing procedures are generally not applicable for the detection of a single pathogen in a complex microbial flora due to limitations of the specificity and sensitivity of the typing procedures. For instance, Salmonella has a high degree of homogeneity with E. coli. Accordingly, a high degree of specificity is required to identify Salmonella in environments in which E. coli is also present. To overcome the specificity limitations of current Salmonella detection procedures, the procedures frequently require that a sample in which Salmonella is to be detected be subjected to Salmonella-specific growth conditions before subjecting the sample to DNA identification methods. (See, Quinn C., Ward J., Griffin M., Yearsley D., Egan J. (1995) The Comparison of a Conventional Culture and Three Rapid Methods for the Detection of Salmonella in Poultry Feeds and Environmental Samples Appl. Microbiol. 20:89-91; Cudjoe K. S., Hagtubeet, T., Dainty R. (1995) Immunomagnetic Separation of Salmonella from Foods and their Detection Using Immunomagnetic Particle (IMP)-ELISA Int. J. Food Microbiol. 27:11-25); and (Meer R. R., Park D. L. (1995) Immunochemical Detection Methods for Salmonella SPP, Escherichia coli O157:87 and Listeria Monocytogenes in Foods Rev. Environmental Contam. Toxicol. 142:1-12).
The Salmonella-specific growth conditions amplify the relative amount of Salmonella within the sample and thereby enrich the sample in Salmonella. However, such Salmonella enrichment procedures undesirably add time and expense to Salmonella detection methods. Accordingly, it would be desirable to develop Salmonella detection methods which could be used to identify Salmonella in a complex microbial flora without requiring Salmonella enrichment procedures. It would further be desirable to develop a method which could detect or recognize a Salmonella pathogen in sample directly obtained from tissues, food materials or the environment without requiring prior selective amplification of Salmonella within the sample.
A PCR-gene-probe based assay has potential for improving routine monitoring of Salmonella (Hanes D. E., Koch W. H., Miliotis M. D., Lampel K. A. (1995) DNA Probe for Detecting Salmonella Enteritidis in Food. Mol. Cellular Probes:9-18). However, more Salmonella-specific determinants have to be discovered before useful PCR-gene-probe assays can be utilized. Accordingly, it would be desirable to identify Salmonella-specific sequences which could be utilized as Salmonella-specific determinants.
Once a Salmonella-specific sequence is identified, a number of in-vitro gene amplification protocols may be utilized for detecting the determinant. Such gene application protocols include: polymerase chain reaction (PCR), ligase chain reaction (LCR), Q.beta. replicase amplifications, 3SR amplifications, and transcription-based amplification systems (TAS). (See, Pillai S. D. and Ricke S. C. (1995) Strategies to Accelerate the Applicability of Gene Amplification Protocols for Pathogen Detection in Meat and Meat Products Crit. Reviews in Microbiology, 21(4):239-261.) While gene amplification approaches have shown some promise, they also have shortcomings which have detracted from their usefulness inasmuch as the existing protocols require the time consuming and costly step of culture enrichment.
Also, once a Salmonella-specific sequence is identified and isolated it is conceivable that it could be attached to a chip to form a DNA chip. This could be subsequently utilized for detecting the presence of the identified Salmonella-specific determinant. (See, Chee M., Yang R., Hubbel E., Berno et al (1996) Accessing Genetic Information With High-Density DNA Arrays Science 274:610-614).
Currently, several efforts are underway to sequence various Salmonella species. For instance, a Salmonella typhimurium sequence, shown below as SEQ ID NO:1, has been identified by Baumler et. al. (See, Baumler A. J., Kusters J. G., Stojiljkovic I., Heffron F. (1994) Salmonella typhimurium Loci Involved in Survival within Macrophages, Infect. Immun. 62:1623-30.)
Other identified sequences of a Salmonella species are described by Wong et. al. (Wong K. K., Wong R. M., Rudd K. E., McClelland M. (1994) High-Resolution Restriction Map for a 240-Kilobase Region Spanning 91 to 96 Minutes on the Salmonella typhimurium LT2 Chromosome Journ. of Bacteriology, p. 5729-5734). Wong et. al. provides a restriction map for a 240-kilobase region of a Salmonella typhimurium LT2 chromosome.
It would be desirable to develop a means by which the genus Salmonella may be readily identified by gene amplification or other procedures, and which avoids the shortcoming attendant to the prior art techniques and practices.
Further, as Salmonella is responsible for much illness throughout the world, it would be desirable to develop procedures which block the virulence of Salmonella.